The cell cycle in eukaryotic organisms is a highly regulated network of processes that contains numerous checks and balances to ensure its proper and timely execution. Cell cycle checkpoints ensure that the events during a specific period of the cell cycle are carried out appropriately before proceeding to the next phase of the cell cycle. The checkpoint mechanisms of interest in this proposal are those that respond to DNA damage and to problems arising during DNA replication. Defects in checkpoints result in increased chromosome instability and such defects are known cancer predisposition conditions in human. DNA damage and DNA replication checkpoint pathways have been conserved from yeast to human which makes the former an ideal organisms for fundamental checkpoint studies. The primary focus of this proposal will be on the sensor protein kinase Mec1, the ortholog of human ATR, that initiates the checkpoint. Mec1/ATR kinase activity is activated as the initiating step of the checkpoint pathway, through interaction with specific activators that show cell-cycle phase specificity. A second protein kinase, Rad53, which is related to human Chk1 and Chk2, acts downstream in halting the cell cycle and activating other response pathways. While numerous genetic and molecular biological studies have identified many of the factors involved in the various checkpoints, the biochemical mechanisms of activation of Mec1 and of Rad53 have remained relatively obscure. The biochemical studies in this proposal are aimed at understanding Mec1 activation by the replication protein Dna2 (Aim 1); to study the structure of Mec1 by cryo-electron microscopy and crystallogaphy and the nature of activation of Mec1 by structure-function analysis using small peptides as probes (Aim 2); and to study the role of DNA binding in activation of Rad53 (Aim 3). These biochemical experiments will be complemented by genetic studies to obtain an integrated view of checkpoint activation.